DESC:FIELD OF INVENTION

The present invention relates to a microbial consortium comprising of two or more microbial strains capable of forming biofilm after coming in contact with moisture containing soil or aqueous media. The present invention further relates to preparing formulations comprising biofilm forming microbial consortia to use as biofertilizer. The present invention also relates to encapsulating the said microbial strains to protect the formulation from inactive excipients of the biochemical fertilizer.

BACKGROUND OF THE INVENTION

Fertilizers are widely used to improve plant and crop growth and address poor soil conditions. The most commonly used fertilizers are inorganic chemical types. However, these chemical fertilizers can be costly to produce, pose safety risks, and often lead to environmental problems, such as nitrate pollution in runoff and groundwater. Reducing the use of chemical fertilizers can help promote environmental sustainability.

Both scientific research and public opinion are increasingly recognizing the value of beneficial microorganisms and their unique properties. These microorganisms play a vital role in agriculture, animal and human health, and waste management. In agriculture, they are used to fertilize plants, improve composting, and enhance soil quality. In both animals and humans, beneficial bacteria help prevent diseases caused by harmful bacteria that disrupt the natural microbial balance. In waste management, microorganisms accelerate the decomposition of waste and break down odorous compounds.

Biofertilizers, which contain microorganisms, are increasingly seen as alternatives to traditional chemical fertilizers. The role of certain bacteria in supporting plant growth has been recognized for a long time. For instance, nitrogen-fixing bacteria like Rhizobium provide essential nitrogen to plants. Other bacteria, such as Azotobacter and Azospirillum, also help boost plant growth and increase crop yields by enhancing nutrient accumulation. However, these bacteria often struggle to compete with the native microorganisms in soil and plants, requiring large amounts of inoculum. Bacillus and Pseudomonas species have also been used in microbial fertilizers.
In agriculture, there is a growing demand to replace or supplement traditional fertilizers with eco-friendly biofertilizers. However, biofertilizers face challenges related to stability, storage, and effectiveness. As a result, there is a need for the development of microorganism-based technologies and products that are stable under various conditions, maintain high concentrations of microbes, have a long shelf life, and are easy to use.

US6228806B1 discloses the Fertilizer compositions comprising: A) an effective quantity of an inorganic or organic fertilizer; and B) a quantity of beneficial microorganisms sufficient to further enhance plant growth when the fertilizer composition is applied to soil and/or control pathogens in the soil, and methods for their use.

US20200337314A1 discloses the Solid form compositions for the treatment of natural bodies of water comprising A) a quantity of beneficial aerobic microorganisms that will control at least one of an organic pollutant, an algae, and a weed in at least a portion of said body of water; and B) a growth accelerator for component A); wherein the solid form composition weighs at least about 85 grams.

IN202117022217 discloses the integration of exogenous microbial biofilms to confer increased stability and viability for an extended shelf life of desired microbes (e.g., bacteria), as compared to those microbes in the absence of the exogenous microbial biofilms. The microbes include transgenic microbes, non-transgenic microbes, and non-intergeneric remodelled microbes. The utilization of the taught microbial products will enable a significant expansion of the typical shelf life of microbial compositions. The microbes comprising exogenous biofilms taught herein are able to be combined with other agriculturally beneficial compositions.

Available the prior art talks about the reduction of biofilm on certain substances but does not disclose the invention with formation of biofilm in soil or any aqueous media. Moreover, no prior art discloses the beneficial use of biofilm formation for environmentally stable use. Further, the prior art does not disclose a microbial consortia protected by polymeric encapsulation used in conjunction with chemical fertilizers. No invention discloses having a consortia with the capacity of forming microbial biofilm. The present invention discloses a composition expected to offer a practical, evidence-supported approach for enhancing the growth and yield of plants, providing a readily accessible method for integrating essential nutrients and microbial consortia into the soil.

SUMMARY OF THE INVENTION

The main aspect of the present invention is a biofilm comprising of microbial consortium selected from group; namely (a) Bacillus group (1.0-40.0% w/w), (b) Pseudomonas group (1- 30% w/w) and (c) Actinomycete group (1-45% w/w).

Another aspect of the present invention provides the process of the formation of biofilm in the presence of moisture, microorganisms detects each other through quorum signalling for further forming a biofilm in the desired area. Microorganisms are grown and each strain sporulated separately, then encapsulate them in a layered protection system and then mix them all together.

Yet another aspect of the present invention is to grow and encapsulate each strain separately. Each individually encapsulated microorganism strain is then packed together which forms a biofilm through the process of quorum signalling, when they come in contact with moisture.

An aspect of the present invention is to protect the microbial consortia with encapsulation which prevents direct exposure of organisms to the fertilizer and are activate only after exposure to the moisture in the soil/aqueous media.

Another aspect of the present invention is to prepare biofertilizer formulations comprising dry inoculants, slurries, granular and liquid formulation.

Yet another aspect of the present invention is to use fertilizer as carrier which ensures the microbes are constantly delivered to maintain the biological activity required to improve soil quality and reduce the use of chemical alternatives.

A further aspect of the present invention is the microbial consortia which reduces the phosphate and nitrate concentration from agricultural runoff. Hence, reducing the pollutant seepage into the soil.

A plant grown in soil mixed with the consortium of the present invention results in a voluminous yield and plant growth with better quality.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Life-Cycle of Biofilm formation
Figure 2: Bio-chemical Composition and CFU on NA after 10 days interval.
Figure 3: Biochemical Composition and CFU on Jensen after 10 days interval.
Figure 4: Crystal violet assay of biofilm formation on micro-titer plate.
Figure 5: Biofilm formation on Tryptone Soyapeptone Agar

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. The present technology is also illustrated by the examples herein, which should not be construed as limiting in any way.

As used herein, the terms below have the meanings indicated. The singular forms "a," "an," and "they" may refer to plural articles unless specifically stated otherwise.

The term "about," as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term "about" should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures.

“Consortia” is two or more bacterial or microbial groups living symbiotically. Consortiums can be endosymbiotic or ectosymbiotic or occasionally may be both.

The term "plant growth-promoting activity", as used herein, encompasses a wide range of improved plant properties, including, for example without limitation, improved nitrogen fixation, improved root development, increased leaf area, increased plant yield, rapid seed germination, increased photosynthesis, or an increased in accumulated biomass of the plant. A microbial consortium of the present invention, supply a plant with nutrients and thereby can promote plant growth.

The “Bacillus subtilis” known also as the hay Bacillus or grass Bacillus, is a gram-positive, catalase positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges.

The “Pseudomonas aeruginosa” is a common encapsulated, gram-negative, aerobic-facultatively anaerobic, rod-shaped bacterium that can cause disease in plants and animals, including humans.

The “Actinomycete” are a diverse phylum of gram-positive bacteria with high guanine and cytosine content.

The term “Biofilms” are mucilaginous communities of microorganisms such as bacteria, archaea, fungi, moulds, algae or protozoa or mixtures thereof that grow on various surfaces. Biofilms form when microorganisms establish themselves on a surface and activate genes involved in producing a matrix that includes exopolysaccharides.

The term “quorum signalling”, a used herein, regulate their cooperative activities and physiological processes through a mechanism called quorum signalling (QS), in which bacterial cells communicate with each other by releasing, sensing and responding to small diffusible signal molecules.

The term “quorum-sensing” signals help trigger and coordinate part of the process of forming a biofilm after the microorganism attaches to the surface. Bacteria constantly secrete low levels of the signals and sense them either through receptors on their surfaces, or internally. The receptors trigger behavioural changes when there are enough bacteria to allow the signals' concentrations to achieve a critical threshold. Once this occurs, bacteria respond by adopting communal behaviour, such as forming a biofilm, and in the case of pathogenic bacteria, deploying virulence factors such as toxins. In addition to communicating with members of their own species, bacteria also conduct inter-species communications, such that a biofilm may contain more than one species of bacteria.

The term “microencapsulation” signals process of coating or enclosing a core material with a polymeric substance to create microspheres ranging in size from 1 to 1000 µm. This adaptable technology has been used to encapsulate a variety of products, including pharmaceuticals, flavours, volatile oils, plant extracts, and enzymes. In recent years, it has also been applied to microbial cell immobilization, offering several advantages over other techniques, such as higher cell loading capacity, improved cell survival, and increased production rates of desired microbial products. By trapping microbial cells within a semi-permeable polymeric matrix, this method physically isolates the cells from the external environment while preserving a favourable internal micro-environment.

Bacillus subtilis strongly affect the environment by increasing nutrient availability to the plants. Bacillus is able to maintain stable contact with higher plants and promote their growth. Bacillus species used as biofertilizers have a direct effect on plant growth through the synthesis of plant growth hormones.

Pseudomonas Species is ubiquitous bacteria in agriculture soils and has many traits that make them well suited as PGPR. The most effective strains of Pseudomonas have been fluorescent Pseudomonas spp. The presence of Pseudomonas fluorescence inoculant in the combination of microbial fertilizer plays an effective role in stimulating yield and growth traits of plants.

Actinomycetes are one of the prominent soil microbes and there are evidences that actinomycetes grow in close association with the plant roots and are one of the important groups of root-colonizing microorganisms. Actinomycetes are important, not only as degraders of organic matter in the natural environment, but also as producers of antibiotics. Many actinomycetes are important source of enzymes, such as chitinase, cellulases, peptidases, proteases, xylanases, ligninases, amylases, pectinases and keratinase. The production of these hydrolytic enzymes make it possible for actinomycetes to break down organic matter in their natural environment.

“Biofilms” are communities of microorganisms that grow attached to a surface or interphase and embedded in a self-produced extracellular matrix. Inside the biofilm, bacteria grow protected from environmental stresses, such as desiccation, attack by the immune system, protozoa ingestion, and antimicrobials. Bacteria built the biofilm comprises three sequential stages: irreversible adhesion to the surface, followed by bacterial division and production of the extracellular matrix and finally, disassembly of the matrix and dispersion of bacteria. when the attached bacteria divide and form micro colonies that the population density increases and quorum signals can reach sufficient levels to activate the maturation and disassembly of the biofilm in a coordinate manner. Biofilm dispersion is essential to allow bacteria to escape and colonize new niches when nutrients and other resources become limited and waste products accumulate. There are different strategies to accomplish biofilm dispersion: ending the synthesis of the biofilm matrix compounds, degrading the matrix and also, disrupting noncovalent interactions between matrix components. Because QS regulatory networks are usually very intricate and may include several genes whose products affect biofilm development at different stages, it is not always easy to understand how the activation of QS finally triggers biofilm dispersion.

Biofilm consortia refer to groups of different bacterial species that come together and form biofilms, which are complex communities of microorganisms embedded within a self-produced matrix of extracellular polymeric substances (EPS). These biofilms can form on a variety of surfaces, including natural environments (like rivers, soil) and artificial surfaces (like medical devices or pipes). The theory behind biofilm consortia bacteria focuses on the interaction, cooperation, and competition between different bacterial species within a biofilm community.

In a biofilm consortium, different species of bacteria can interact in various ways. Some may have mutualistic relationships, where both benefit from the association (e.g., nutrient exchange or protection from environmental stress). Others might have commensal relationships, where one species benefits without affecting the other, or competitive interactions, where species compete for limited resources like space, oxygen, or nutrients.

One of the central ideas behind biofilm consortia is the division of labor. Different species may specialize in different functions that contribute to the survival of the entire biofilm. For example:
• Some bacteria might specialize in degrading a particular compound or nutrient that is inaccessible to others.
• Some species could secrete enzymes to break down organic matter, while others might help in producing EPS, which acts as a scaffold to hold the biofilm together.

In a biofilm consortia, the different bacteria exchange nutrients and metabolites. For example, in a multispecies biofilm consortia, one bacterium may degrade a complex molecule, releasing smaller molecules that other bacteria can use as nutrients. This metabolic cooperation makes biofilms more efficient in terms of nutrient acquisition compared to single-species biofilms.

Figure 1 depicts the life-cycle of biofilm formation. The lifecycle include 4 stages mainly adhesion, microcolony formation, maturation and dispersion.

The biofilm matrix, made up of extracellular polymeric substances (such as polysaccharides, proteins, and DNA), offers a physical barrier that protects the bacteria from environmental stressors, such as changes in pH, temperature, antibiotics, or immune system attacks. In consortia, this protection can be amplified, as different species contribute to the overall structure and resilience of the biofilm. Some species may also secrete antimicrobial compounds that deter competitors or predators.

In microbiology, a colony-forming unit (CFU, cfu or Cfu) is a unit which estimates the number of microbial cells (bacteria, fungi, viruses etc.) in a sample that are viable, able to multiply via binary fission under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies, it is uncertain if the colony arose from a single cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.

Nutrient agar medium is used for maintaining microorganisms, cultivating fastidious organisms by enriching with serum or blood and are also used for purity checking prior to biochemical or serological testing (Figure 2).

Jensen’s Medium (Agar) is used for detection and cultivation of nitrogen fixing bacteria. These bacteria such as Rhizobium species utilize nitrogen from the atmosphere for their cell protein synthesis. This cell protein is then mineralized in soil after the death of the cells thereby contributing towards the nitrogen availability of the crop plants (Figure 3).

“Horizontal gene transfer (HGT)”, also known as lateral gene transfer (LGT), refers to the transfer of genetic material between organisms, distinct from the vertical transmission of DNA from parent to offspring during reproduction. HGT plays a crucial role in the evolution of various organisms, significantly shaping our understanding of higher-order evolution, especially in bacterial development.

HGT is the main mechanism through which antibiotic resistance spreads in bacteria, and it also contributes to the evolution of bacteria capable of breaking down novel substances like human-made pesticides. Additionally, it aids in the evolution, maintenance, and spread of virulence. HGT often involves elements like temperate bacteriophages and plasmids. Antibiotic resistance genes from one bacterial species can be transferred to another via HGT processes such as transformation, transduction, and conjugation, enabling the recipient bacteria to resist antibiotics. The rapid spread of antibiotic resistance in this way poses a growing challenge in medical practice. Ecological factors may also influence the transfer of antibiotic-resistant genes.

The main aspect of the present invention provides a formulation of microbial consortia; namely Bacillus group (1-40%), Pseudomonas group (1- 30%) and Actinomycete group (1-45%). This formulated consortia helps to form a biofilm when comes in contact with any kind of moisture contain in soil/ water and suited to influence soil conditions and plant growth.

The Bacillus sp. used in the microbial consortia is selected from the Bacillus subtilis Bacillus coagulans; Bacillus sphericus; Bacillus megaterium; Bacillus thurirgensis; Bacillus steareothermophilus; Bacillus polymyxa; Bacillus cereus; Bacillus globigi; Bacillus halodurans Bacillus azotofixans; Bacillus azotoformans; Bacillus larvae; Bacillus lentimorbus; Bacillus popilliae; Bacillus sphaericus; Bacillus licheniformis; Clostridium difficile; Bacillus paramycoides; Bacillus amyloliquefaiens; Bacillus nitratireducens; LyciniBacillus fusiformis; AberriniBacillus migulanus; Clostridium tetani; Clostridium botulinum; Clostridium perfringens; Clostridium perfringens.

The Pseudomonas sp. used in the microbial consortia is selected from the Azotobacter sp.; Pseudomonas flourescens; Pseudomonas aureofaciens; Saccharomyces cerevisiae; Arthrobacter sp.; Flavobacterium sp.; Streptomyces sp.; Aspergillus sp.; Trichoderma sp; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pseudomonas putida; Pseudomonas cepacia; Pseudomonas stutzeri; Pseudomonas maltophilia; and Pseudomonas putrefaciens.

The Actinomycetes used in the microbial consortia is selected from the Actinomyces viscosus; Actinomyces meyeri; Actinomyces naeslundii; Actinomyces odontolyticus; Actinomyces gerencseriae; Actinomyces neuii; Actinomyces turicensis; Actinomyces radingae; Actinomyces israelii; Actinomyces gerencseriae;

Diffusion-Controlled Release: The biofilm matrix has a porous structure that allows the slow diffusion of nutrients, moisture, or microbial cells. When the external environmental conditions (like moisture content, pH, or temperature) change, the release rate of beneficial microorganisms from the biofilm matrix is regulated. The microorganisms are slowly released when they encounter conditions in the soil that support microbial growth, such as a nutrient-rich environment.

Matrix Degradation: The EPS matrix of the biofilm can degrade over time due to the activity of microorganisms or external factors like soil enzymes, moisture, or temperature changes. This breakdown results in the gradual release of microorganisms or microbial metabolites, which can enhance nutrient cycling and plant growth. The microbial consortia is designed to degrade slowly over weeks or months, releasing nutrients and microbes in synchronization with plant growth cycles.

Microbial Activity-Induced Release: The activity of microbes within the biofilm can also influence the controlled release of nutrients or growth-promoting substances. For instance, certain biofilm-forming bacteria can produce extracellular enzymes that break down organic matter, releasing nitrogen, phosphorus, or other essential nutrients for the plant. Nitrogen-fixing bacteria in the biofilm gradually release nitrogen into the soil over time, promoting sustainable fertilization.

Environmental Stimuli: The biofilm consortium can be engineered to release microorganisms in response to specific environmental cues (e.g., pH change, temperature, or salinity). This can help synchronize the release with the plant’s nutrient demands or growth stages. A biofilm that releases phosphorus-solubilizing bacteria when soil phosphorus levels are low would provide nutrients exactly when the plant needs them.

The microorganisms present in the formulation of microbial consortia, Bacillus subtilis produce and release chemical signal molecules which in return increases in concentration of a cell density in order to detect the Pseudomonas species present in the microbial consortia to form the biofilm in the soil/aqueous media.

The microorganisms present in the formulation of microbial consortia, Pseudomonas species produce and release chemical signal molecules which in return increases in concentration of a cell density in order to detect the actinomycetes species present in the microbial consortia to form the biofilm in the soil/aqueous media.

The microorganisms present in the formulation of microbial consortia, Bacillus species produce and release chemical signal molecules which in return increases in concentration of a cell density in order to detect the actinomycetes species present in the microbial consortia to form the biofilm in the soil/aqueous media.

The resultant biofilm matrix comprises extracellular polymeric substances such as polysaccharides, proteins and debris of genetic material, offering physical barrier that protects the microbes from environmental stressors. In the present consortia, this protection is enhanced as different species of microbes are contributing to the biofilm formation. Actinomycetes present in the said consortia produces secondary metabolites with biopesticide properties which inhibit infestation in crops.

Additionally, Pseudomonas secondary metabolites exhibit antibiotic properties as well as nitrogen fixing properties further contributing to the synergism of the microbial consortia by providing the protection to the said consortia while improving the quality of soil. Furthermore, Bacillus strain has the ability to survive in extreme conditions such as high temperatures and high salt conditions in addition to aiding the plant with nutrient absorption, improved immunity from plant pathogens and nitrogen fixing properties.

Hence, another aspect of the present invention is to provide the microbial consortia an ideal environment favourable for horizontal gene transfer (HGT) between the member strains for the said consortia. Thus, over time each member of the microbial consortia develops new properties acquired from other microbial stains present in the said consortia further enhancing the resultant biofertilizer.

Moreover, the microbes of the said consortia are microencapsulated within a polymeric layer to protect them from environmental exposure. A major aspect of the present invention is to encapsulate the mixture of all three microbial strains together. This mixture is then able to form a biofilm after being exposed to moisture.

A further aspect of the present invention is to encapsulate each microbe separately and then packed together. This process of encapsulation protects the microbial integrity of each strain until it is exposed to moisture upon its release on to the soil resulting in the biofilm formation.

In an embodiment, provides a formulation of microbial consortium that serve as carriers to deliver minimum set of organisms to the soil found to be exceptionally well suited to influence soil conditions and plant growth promotion. Using fertilizer as carriers will ensure that the microbes are constantly delivered to maintain the biological activity required to improved soil productivity and reduce chemical inputs.

An embodiment of the present invention, provides a formulation of microbial consortia comprising (a) Bacilli group (1-40%), (b) Pseudomonas group (1- 30%) and (c) Actinomycete group (1-45%). Firstly, the microbes are encapsulated to prevent the direct exposure of organisms to the fertilizer and are activate only after exposure to the moisture in the soil/aqueous media. Then, the encapsulated microbes blended with a ribbon blender to form a dry powder form. Lastly, the dry powder form is dusted on the granules of the fertilizer.

In an embodiment of the present invention, biofilm development and quorum sensing are described as closely interconnected processes. Biofilm formation is a cooperative group behaviour that involves bacterial populations living embedded in a self-produced extracellular matrix. Quorum sensing (QS) is a cell-cell communication mechanism that synchronizes gene expression in response to population cell density. Bacteria are elementary, unicellular organisms able to grow, divide, sense and adapt to environmental signals autonomously. Despite their self-sufficiency, bacteria coordinate efforts with neighbours to accomplish cooperative activities such as bioluminescence production, biofilm development, and isozyme secretion. Coordination occurs through a mechanism of cell-to-cell communication called quorum sensing (QS). QS provides bacteria the capacity to recognize the population density by measuring the accumulation of a specific signalling molecule that members of the community secrete. Only when the population density is high, the accumulation of the signal in the extracellular environment is sufficient to activate the response. Structurally, QS signal molecules have a low molecular weight and belong to a wide range of chemical classes including acyl homoserine lactones (AHLs), furanosyl borate diesters (AI2), cis-unsaturated fatty acids (DSF family signals) and peptides.

Pseudomonas aeruginosa
Biofilm formation has been extensively studied in the Gram negative bacterium P. aeruginosa because of its implication in causing severe chronic infections in patients with cystic fibrosis. P. aeruginosa harbours two complete AHL circuits, LasI/LasR and RhlI/RhlR, being the LasI/R circuit hierarchically positioned upstream the RhlI/R circuit.

These two QS systems are composed of a LuxI type synthase, responsible of AHL synthesis, and a LuxR type receptor. At high cell density (HCD), AHLs accumulate and specifically interact with LuxR type transcription factors. AHL binding stabilizes the LuxR type proteins, allowing them to fold, bind DNA, and regulate transcription of target genes. AHL bound LuxR type proteins also activate transcription of luxI, providing a signal amplification mechanism via a feed forward auto induction loop. P. aeruginosa has two orphan LuxR homologues, VqsR and QscR, and it also presents the Pseudomonas quinolone signal (PQS), which are interconnected with the LasI/LasR and RhlI/RhlR signalling circuitries.

In yet another embodiment of the present invention is to provide formulation of biofertilizer selected from granular, liquid, dry inoculants and slurries.

Further embodiment of the present invention is to provide microbial consortium Granule (GR) formulation comprising formulation excipients from the category of Wetting cum emulsifying agent, Dispersing agent, solvent, carrier, colouring agent, controlled release agent, preservative and stabiliser.

In an embodiment wetting cum emulsifying agent for the present Granule (GR) is selected from and not limited to Mono C2-6 alkyl ether of a poly C2-4alkylene oxide block copolymer, condensation product of castor oil and polyC2-4alkylene oxide, alkoxylated castor oil is available under the trade name Agnique CSO-36, a mono- or di-ester of a C12-24 fatty acid and polyC2-4alkylene oxide, carboxylates, sulphates, sulphonates, alcohol ethoxylates, alkyl phenol ethoxylates, fatty acid ethoxylates, sorbitan esters, ethoxylated fats or oils, amine ethoxylates, phosphate esters, ethylene oxide - propylene oxide copolymers, fluorocarbons,alkyd-polyethylene glycol resin, polyalkylene glycol ether, apolyalkoxylated nonyl phenyl, alkoxylated primary alcohol, ethoxylated distyrylphenol, ethoxylated distyrylphenol sulphate, ethoxylated tristyrylphenol phosphate, tristyrylphenol phosphate ester, hydroxylated stearic acid polyalkylene glycol polymer, and their corresponding salts, alkyd-polyethylene glycol resin, polyalkylene glycol ether, ethoxylated distyrylphenol, ethoxylated distyrylphenol sulphate, ethoxylated tristyrylphenol phosphate, Alkyl phenol ethoxylate and ethoxylated tristrylphenol phosphate blend, tristyrylphenol phosphate ester, tristyrylphenol phosphate potassium salt, dodecysulfate sodium salt.

In an embodiment Dispersing agent for the present Granule (GR) is selected from Copolymer of propylene oxide (PO) and ethylene oxide (EO) and/or an ethoxylated tristyrene phenol, copolymer of PO and EO is alpha-butyl-omega-hydroxypoly (oxypropylene) block polymer with poly(oxyethylene), ethoxylated tristyrene phenol is alpha-[2,4,6-tris[1-(phenyl)ethyl] phenyl]-omega-hydroxy poly (oxyethylene, poly(oxy-1,2-ethanediyl)-alpha-C10-15alkyl-omega-hydroxy phosphate or sulphate and/or a C10-13 alkylbenzenesulfonic acid, tristyrylphenols, nonylphenols, dinonylphenol and octylphenols, styrylphenol polyethoxyester phosphate, alkoxylated C14-20 fatty amines.

In an embodiment Solvent for the present Granule (GR) formulation is selected from Water, n-Octanol, n-butanol, Propylene glycol, polypropylene glycol, Fatty acid methyl ester, cyclohexane, xylene, mineral oil or kerosene, mixtures or substituted naphthalenes, mixtures of mono- and polyalkylated aromatics, dibutyl phthalate or dioctyl phthalate, ethylene glycol monomethyl or monoethyl ether, butyrolactone, octanol, castor oil, soybean oil, cottonseed oil, epoxidised coconut oil or soybean oil, aromatic hydrocarbons, dipropyleneglycol monomethylether, polypropylene glycol, polyoxyethylene polyoxypropylene glycols, polyoxypropylene polyoxyethylene glycols, diethyleneglycol, polyethylene glycol, methoxy polyethylene glycols; glycerol, methyl oleate, n-octanol, alkyl phosphates such as tri-n-butyl phosphate, propylene carbonate and isoparaffinic, tetrahydrofurfuryl alcohol, gamma-butyrolactone, N-methyl-2-pyrrolidone, tetramethylurea, dimethylsulfoxide, N,N-dimethylacetamide , Diacetone alcohol, Polybutene, Propylene carbonate, Dipropylene glycol isomer mixture.

Preservative for the present Granule (GR) formulation is selected from and not limited to 1,2 benzisothiazolin-3-one, 2-Methyl-2H-isothiazol-3-one, 5-chloro-2-methyl-4isothiazolin-3-one or sodium benzoate or benzoic acid.
Stabilizer for the present Granule (GR) is selected from and not limited to carboxylic acids (citric acid, acetic acid), orthophosphoric acid dodecyl benzene sulfonic acid and salt thereof.

In an embodiment, Carrier for the present Granule (GR) formulation is selected from Diatomaceous earth, attapulgite or zeolites, dolomite, limestone, silica, fly ash, hydrated lime, wheat flour, wood flour, ground wheat straw, cellulose and soy flour, bentonite, kaolin, attapulgite, diatomaceous earth, calcium carbonate, talc, muscovite mica, fused sodium potassium, aluminum silicate, perlite, talc and muscovite mica, urea, sulfur-coated urea, isobutylidene diurea, ammonium nitrate, ammonium sulfate, ammonium phosphate, triple super phosphate, phosphoric acid, potassium sulfate, potassium nitrate, potassium metaphosphate, potassium chloride, dipotassium carbonate, potassium oxide and a combination of these. Calcium, magnesium, sulfur, iron, manganese, copper, zinc; oxides, humic acid, Wood floor, Calcium silicate, Cellulose granules, Magnesium stearate.

In an embodiment, Colouring agent for the present Granule (GR) is selected from Crystal violet, Thalocyano dye chlorinated, Aerosol green FFB dye, Rodamine, Azo compound.

In an embodiment, Controlled release agent for the present Controlled Release granule (CR-GR) formulation is selected from and not limited to Xanthan gum, PVK, carboxy methyl celluloses, polyvinyl alcohols, gelatin, sodium carboxy methyl ellulose, hydroxyl ethyl cellulose, Sodium Polyacrylate, modified starch, Paraffin wax, Polyvinyl acetate, Montan wax and vinyl acetate, Polyethylene Glycol, Cationic hydro soluble polymer, C4 alkylated Polyvinyl pyrrolidone.

Microorganisms are grown and sporulated each strain separately, then encapsulate them in a layered protection system and then mix them all together. The process of biofilm formation in present invention begins with the dissolution of the encapsulation polymer once it comes in contact with any kind of moisture contained in soil or aqueous media. Once the encapsulation layer is dissolved, the individual microbial strains grow by utilizing the nutrients from the soil and begin the formation of a biofilm once they come in contact with other aforementioned strains through the process of quorum signalling.

The consortia is protected with encapsulation which prevents direct exposure of organisms to the fertilizer and are activate only after exposure to the moisture in the soil.

In yet another embodiment of the present invention, the microbial strains are encapsulated separately and mixed with the inactive excipients before releasing into the soil.

In an embodiment, provides a consortia in which choice of microorganism is based on the natural spore forming capacity of Bacilli to ensure long stable shelf life despite mixed with chemical fertilizer and despite atmospheric conditions.

Examples:

Example 1: Preparation of Microbial Consortia – T1 (Bacillus subtilis + Pseudomonas florescence + Strptomyces hygroscopicus)

The Bacillus subtilis was acquired from soil samples from Bhuj, Gujarat, Pseudomonas florescence and Strptomyces hygroscopicus was acquired from Maharaja Sayajirao University of Baroda. All three bacterial species were serially diluted and streaked on Nutrient Agar plates separately. Streaked plates were incubated for 24h at 37 °C. All the strains were screened morphologically based on their appearance and isolated to be co-cultured in liquid broth medium. All three bacterial species were individually inoculated into 1000mL Nutrient Broth. The broths were incubated for 24h at room temperature on a rotary shaker. All the cultures were stored at 4 °C under sterile condition. Finally, inoculum was collected from each of the liquid cultures were collected and inoculated into a new Nutrient broth liquid medium in a ratio 2:3:3 to prepare 200ml of microbial consortia.

Example 2: Preparation of Microbial Consortia – T2 (Bacillus thurirgensis + Bacillus sphaericus + Pseudomonas aureofaciens + Actinomyces turicensis)

The Bacillus thurirgensis, Bacillus sphaericus was acquired from soil samples from Gujarat, Pseudomonas aureofaciens and Actinomyces turicensis was acquired from Maharaja Sayajirao University of Baroda. All three bacterial species were serially diluted and streaked on Nutrient Agar plates separately. Streaked plates were incubated for 24h at 37 °C. All the strains were screened morphologically based on their appearance and isolated to be co-cultured in liquid broth medium. All three bacterial species were individually inoculated into 1000mL Nutrient Broth. The broths were incubated for 24h at room temperature on a rotary shaker. All the cultures were stored at 4 °C under sterile condition. Finally, inoculum was collected from each of the liquid cultures were collected and inoculated into a new Nutrient broth liquid medium in a ratio of 1:2:4:3 to prepare 200ml of microbial consortia.

Example 3: Preparation of Microbial Consortia – T3 (Bacillus thurirgensis + Bacillus sphaericus + Bacillus coagulans + Bacillus steareothermophilus)

The Bacillus thurirgensis Bacillus sphaericus, Bacillus coagulans, and Bacillus steareothermophilus was acquired from soil samples from Gujarat. All three bacterial species were serially diluted and streaked on Nutrient Agar plates separately. Streaked plates were incubated for 24h at 37 °C. All the strains were screened morphologically based on their appearance and isolated to be co-cultured in liquid broth medium. All three bacterial species were individually inoculated into 1000mL Nutrient Broth. The broths were incubated for 24h at room temperature on a rotary shaker. All the cultures were stored at 4 °C under sterile condition. Finally, inoculum was collected from each of the liquid cultures were collected and inoculated into a new Nutrient broth liquid medium in a ratio of 1:1:1:1 to prepare 200ml of microbial consortia.

Example 4: Preparation of Microbial Consortia – T4 (Pseudomonas aureofaciens + Saccharomyces cerevisiae + Pseudomonas putida + Pseudomonas cepacia)

The Pseudomonas aureofaciens, Saccharomyces cerevisiae, Pseudomonas putida, Pseudomonas cepacia were acquired from soil samples from Maharaja Sayajirao University of Baroda. All four bacterial species were serially diluted and streaked on Nutrient Agar plates separately. Streaked plates were incubated for 24h at 37 °C. All the strains were screened morphologically based on their appearance and isolated to be co-cultured in liquid broth medium. All three bacterial species were individually inoculated into 1000mL Nutrient Broth. The broths were incubated for 24h at room temperature on a rotary shaker. All the cultures were stored at 4 °C under sterile condition. Finally, inoculum was collected from each of the liquid cultures were collected and inoculated into a new Nutrient broth liquid medium in a ratio of 1:3:2:2 to prepare 200ml of microbial consortia.

Example 5: Preparation of Microbial Consortia – T5 (Actinomyces gerencseriae + Actinomyces neuii + Actinomyces radingae + Actinomyces israelii)

The Actinomyces gerencseriae, Actinomyces neuii, Actinomyces radingae, and Actinomyces israelii were acquired from soil samples from Karnataka. All four bacterial species were serially diluted and streaked on Nutrient Agar plates separately. Streaked plates were incubated for 24h at 37 °C. All the strains were screened morphologically based on their appearance and isolated to be co-cultured in liquid broth medium. All three bacterial species were individually inoculated into 1000mL Nutrient Broth. The broths were incubated for 24h at room temperature on a rotary shaker. All the cultures were stored at 4 °C under sterile condition. Finally, inoculum was collected from each of the liquid cultures were collected and inoculated into a new Nutrient broth liquid medium in a ratio of 1:4:2:3 to prepare 200ml of microbial consortia.

Example 6: Preparation of Microbial Consortia – T6 (T3 + T4 + T5)
The microbial consortia prepared in the aforementioned examples were mixed together to form another multi-species consortia. All bacterial species were serially diluted and streaked on Nutrient Agar plates separately. Streaked plates were incubated for 24h at 37 °C. All the strains were screened morphologically based on their appearance and isolated to be co-cultured in liquid broth medium. All three bacterial species were individually inoculated into 1000mL Nutrient Broth. The broths were incubated for 24h at room temperature on a rotary shaker. All the cultures were stored at 4 °C under sterile condition. Finally, inoculum was collected from each of the liquid cultures were collected and inoculated into a new Nutrient broth liquid medium in a ratio of 3:6:1 to prepare 200ml of microbial consortia.

Example 7: Biofilm Formation in different Microbial Consortia

Tryptone Soyapeptone Agar is used to co-culture the bacterial strains grown in the nutrient broth and to facilitate the formation of the biofilm. All microbial consortia (T1-T6) were separately plated on the Tryptone Soy agar to check the formation of biofilm.

Microbial Consortia (200ml) Bacillus sp. (ml/consortia) Pseudomonas sp.(ml/consortia) Actinomycetes sp. (ml/consortia) Biofilm Formation
T1 50 75 75 Strong
T2 60 80 60 Strong
T3 200 - - Weak
T4 - 200 - Weak
T5 - - 200 Weak
T6 60 120 20 Very Strong

Example 8: Colony Forming Unit (CFU) Measurement Nutrient Agar plates

All the bio chemical composition with sterile soil were taken after an interval of 10 days for measurement of viability and growth of microbes present in platinum. The count measurement was performed by the method mentioned in fertiliser control order 1985 on NA agar plates 10 days interval upto 60 days.

Table 1. Bio-chemical Composition and CFU on NA after respective days.
Sr no Composition Name Composition(g)/Sterile soil(g)/sterile water(ml) Composition/Sterile soil/sterile water ratio CFU on NA /g After 10
days
CFU on NA /g After 20
CFU on NA /g After 30
CFU on NA /g After 40
CFU on NA /g After 50
CFU on NA /g After 60

1 Control-Sterile
Soil only 0/150g/25ml 1:30:5 0 0 6x103 5x106 1x107 1 x 108
2 Microbial Consortia Only 5gm/150g/25
ml 1:30:5 5.4x108 1x108 3x108 3.4x108 1x106 6 x 109
3 Urea + Microbial Consortia 5gm/150g/25
ml 1:30:5 5x103 8x107 1x105 3x106 1x105 1 x 105
4 MOP + Microbial Consortia 5gm/150g/25
ml 1:30:5 1.9x108 1x108 1x106 5x105 2x106 5 x 106
5 SSP + Microbial Consortia 5gm/150g/25
ml 1:30:5 4.5x104 5x106 6x103 1x105 3x106 2 x 108

Example 9: Colony Forming Unit (CFU) Measurement on Jensen agar plate
All the bio chemical composition with sterile soil were taken after an interval of 10 days for measurement of viability and growth of microbes present in platinum. The count measurement was performed by the method mentioned in fertiliser control order 1985 on Jensen agar plates 10 days interval upto 60 days.

Table 2. Biochemical Composition and CFU on Jensen after 10 days Interval.
Sr. no Composition Name Composition(g)/ Sterile soil(g)/sterile water(ml) Composition/
Sterile soil/sterile water ratio CFU on Jensen /g
After 40 days incubation at RT CFU on Jensen /g
After 50 days incubation at RT CFU on Jensen /g
After 60 days incubation at RT
1 Control-Sterile Soil only 0/150g/25ml 1:30:5 1 x 106 1 x 107 6 x 107
2 Microbial Consortia Only 5gm/150g/25ml 1:30:5 1 x 108 1 x 108 8 x 109
3 Urea + Microbial Consortia 5gm/150g/25ml 1:30:5 1 x 108 1 x 105 1 x 107
4 MOP + Microbial Consortia 5gm/150g/25ml 1:30:5 4 x 104 1 x 104 1 x 107
5 SSP + Microbial Consortia 5gm/150g/25ml 1:30:5 - 1 x 105 1.8 x 106

Example 10: Biofilm Formation (Agar assay)

Biofilm formation is the ability of organisms adhering to abiotic or biotic surfaces to undergo a genetic and physiological switch to form a microbial community encased within an exopolymeric matrix that provides protection from environmental stressors.

Figure 5 shows the results of Congo red binding agar assay, wherein the sample was directly streaked on Modified Cong Red Agar plate. After incubation at 37°C, PG-2, SHAP-7, Platinum and PWS showed characteristic black precipitated colonies while Diamond and Gold showed red colonies. The black precipitation is interpreted as strong biofilm formers wherein, the biofilm comprises primarily of polysaccharide components. Red coloured colonies are typically interpreted as non-biofilm formers.

Example 11: Biofilm formation (Microtiter plate assay)

Figure 4 shows the results of Crystal violet assay of biofilm formation on microtiter plate, wherein the sample was prepared and incubated in Tryptone Soya peptone Agar for 20 h. Planktonic growth was transferred to fresh tubes and measured spectrophotometrically. Microbial growth forming biofilm on microtiter surface was washed and stained with crystal violet dye for 20 min. Excess dye was washed off and that bound to biofilm was dissolved using DMSO and its absorbance measured spectrophotometrically.

Sample showing the best ability to form biofilm upon microtiter surface is GOLD as all cells preferred to be in biofilm mode rather than planktonic. Gold, PG-2, SHAP-7, Platinum and PWS all show strong biofilm formation abilities. However, the biofilm constituents of PG-2, Platinum, PWS and SHAP-7 are likely to have more polysaccharide components in comparison to Diamond and Gold which may be proteinaceous in nature based on Congo Red Agar data. Crystal violet Binding assay is considered a more reliable assay for biofilm formation than Congo red agar assay. Figure 4 depicts the formation of biofilm when microbes are encapsulated in 6 different microencapsulation polymers. Platinum encapsulated microbial consortia showed the highest biofilm forming and exopolysaccharide producing ability.

Example 12: Microbial Consortia mixed with urea

Formation of biofilm comprising of microbial consortium which is helpful for the plant growth is used by mixing with the fertilizer when applied to a soil. The viability of microorganisms when mixed with the fertilizer is described in the below example:

The platinum coated urea is used with the microorganisms. The microbial consortia and various chemical fertilizers were mixed and incubated at room temperature for 10 days. The Bio-chemical Mixture composition was prepared in a ratio of 1:99. 50 gm microbial consortia was mixed with 4950 gm platinum coated chemical fertiliser (Urea) and stored for 10 days at room temperature. This mixture was further used for testing and verification.

Example 13: Microbial consortia mixed with Murate of Potash

The platinum coated Murate of potash (MOP) fertilizer is used with the microorganisms. The microbial consortia and a chemical fertilizer was mixed and incubated at room temperature for 10 days. The Bio-chemical Mixture composition was prepared in a ratio of 1:99. 50 gm microbial consortia was mixed with 4950 gm Platinum coated chemical fertilizer (MOP) and stored for 10 days at room temperature. This mixture was further used for testing and verification.

Example 14: Microbial consortia mixed with Single super phosphate (SSP) fertilizer

The platinum coated Single super phosphate (SSP) fertilizer is used with the microorganisms. The microbial consortia and a chemical fertilizer was mixed and incubated at room temperature for 10 days. The Bio-chemical Mixture composition was prepared in a ratio of 1:99. 50 gm Microbial consortia was mixed with 4950 gm Platinum coated chemical fertilizer (SSP) and stored for 10 days at room temperature. This mixture was further used for testing and verification.

Example 15: Starch encapsulated Controlled Release biofertilizer

The aforementioned microbial consortia is encapsulated in a biodegradable starch polymer. Clay or silica based carriers act as support for biofilms, creating a physical barrier that helps release microorganisms in a controlled manner. Starch helps hold water and nutrients, allowing for slow diffusion into the surrounding soil. Hydrogels, capable of absorbing significant amounts of water, are also utilized in controlled-release biofertilizers. The hydrogel matrix gradually releases the biofilm and its nutrients over time by absorbing and retaining moisture from the soil.

Example 16: Chitosan encapsulated Controlled Release biofertilizer

The microbial consortia is encapsulated in a biodegradable chitosan polymer. Clay or silica based carriers act as support for biofilms, creating a physical barrier that helps release microorganisms in a controlled manner. Chitosan helps hold water and nutrients, allowing for slow diffusion into the surrounding soil. Hydrogels, capable of absorbing significant amounts of water, are also utilized in controlled-release biofertilizers. The hydrogel matrix gradually releases the biofilm and its nutrients over time by absorbing and retaining moisture from the soil.

Example 17: Alginate encapsulated Controlled Release biofertilizer

The microbial consortia is encapsulated in a biodegradable Alginate polymer. Clay or silica based carriers act as support for biofilms, creating a physical barrier that helps release microorganisms in a controlled manner. Alginate helps hold water and nutrients, allowing for slow diffusion into the surrounding soil. Hydrogels, capable of absorbing significant amounts of water, are also utilized in controlled-release biofertilizers. The hydrogel matrix gradually releases the biofilm and its nutrients over time by absorbing and retaining moisture from the soil.

Biological Experiments:

Experiment 1: Improved Plant germination (Cane Plant)
The aforementioned microbial consortia has shown improved germination rate in sugarcane plants. The multi-species microbial consortia (Bio NPK Kit) used along with recommended dose of fertilizer showed improved germination rate of the cane plant by 31%. The multi-species microbial consortia showed a 6% enhanced germination rate with respect to chemical fertilizer alone. Moreover, the multispecies microbial consortia showed higher rate of germination with respect to single-species biofertilizer. The tiller population per hectare is exponentially improved over a 4-month period.

Experiment 2: Cotton Plant yield
Overall cotton yield is improved significantly when control is mixed with bio-product C (comprising microbial consortia). Highest yield is achieved when 80% recommended dose of fertilizer is mixed with bio-product C. A 27.5% increase in yield is seen when the said mixture is compared to control alone. In addition to the yield, plant height and number of boll per plant also improved significantly upon application of the said mixture.
Bio-products:
1. Bio-products A: Diamond (Biofetilizer 1) + Platinum (Biofertilizer 2)
2. Bio-products B: Dharamrut (Biofertilizer 3) + Diamond (Biofetilizer 1) + Platinum (Biofertilizer 2)
3. Bio-products C: Dharamrut (Biofertilizer 3) + Microbial consortia

Experiment 3: Analyzing Soil Health after Application
To analyse the soil health after the application of fertilizer, Nitrogen (N), Potassium (K) and Phosphorus (P) content of the soil was calculated. It was found that when microbial consortia was used with 60% recommended dose of fertilizer, the NPK concentration in the sol is preserved. On the contrary, when various combinations of fertilizer products were applied to the soil, the NPK concentration of the soil was significantly depleted.

,CLAIMS:We Claim,

1. A microbe-based biofilm forming consortia comprising:
a. a microorganism consortia in culture medium using two or more microorganism wherein the said microorganisms are co-cultured, and the microorganisms in the consortium are selected from a group of:
i. microbe (A) a nitrogen fixing bacteria;
ii. microbe (B) an antimicrobial secondary metabolite producing microbe;
iii. microbe (C) a microbe capable of metal chelation;
b. co-culturing aforementioned groups of microbes resulting in a strong biofilm formation;
c. a carbon source; and
d. in-active excipients.

2. The microbe-based biofilm forming consortia as claimed in claim 1, wherein the said microbial consortia for multiple strains of each microbe (A, B and C) was prepared and mixed together to form a multispecies consortia used to form a strong biofilm.

3. The microbe-based biofilm forming consortia as claimed in claim 1, wherein the said microbial consortia converts:
a. atmospheric nitrogen into ammonia in soil;
b. produces secondary metabolite to control the growth of weeds and pest; and
c. produce siderphores for heavy-metal chelation.
wherein, the biofilm acts as physical barrier, and protecting the said consortia from the environmental stresses.

4. The microbe-based biofilm forming consortia as claimed in claim 1, wherein a formulation of biofilm forming microbial consortia is selected from dry inoculants, slurries, granular formulation or liquid formulation.

5. The microbe-based biofilm forming consortia as claimed in claim 1, wherein the said carbon source is selected from molasses, blood meal, kelp meal, cottonseed meal, casein meal and tryptone soy broth.

6. The microbe-based biofilm forming consortia as claimed in claim 1, wherein the inactive excipients are selected from Wetting cum emulsifying agent, Dispersing agent, solvent, carrier, colouring agent, controlled release agent, preservative agent and stabiliser.

7. The microbe-based biofilm forming consortia as claimed in claim 1, wherein the consortium comprises:
a. Microbe (A) a nitrogen fixing bacterial species in the amount of 1.0% to 40.0% w/w;
b. Microbe (B) a metal chelating microorganism in the amount of 1.0% to 30.0% w/w;
c. Microbe (C) an antimicrobial secondary metabolite producing microbial species in the amount of 1.0% to 45.0%.

8. The microbe-based biofilm forming consortia as claimed in claim 1, wherein a nitrogen fixing bacteria is Bacilli sp. is selected from Bacillus coagulans; Bacillus sphericus; Bacillus megaterium; Bacillus thurirgensis; Bacillus steareothermophilus; Bacillus polymyxa; Bacillus cereus; Bacillus globigi; Bacillus halodurans Bacillus azotofixans; Bacillus azotoformans; Bacillus larvae; Bacillus lentimorbus; Bacillus popilliae; Bacillus sphaericus; Bacillus licheniformis; Clostridium difficile; Bacillus paramycoides; Bacillus amyloliquefaiens; Bacillus nitratireducens; LyciniBacillus fusiformis; AberriniBacillus migulanus; Clostridium tetani; Clostridium botulinum; Clostridium perfringens; Clostridium perfringens.

9. The microbe-based biofilm forming consortia as claimed in claim 1, wherein the metal chelating microbe is a Pseudomonas sp. selected from Azotobacter sp.; Pseudomonas flourescens; Pseudomonas aureofaciens; Saccharomyces cerevisiae; Arthrobacter sp.; Flavobacterium sp.; Streptomyces sp.; Aspergillus sp.; Trichoderma sp; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pseudomonas putida; Pseudomonas cepacia; Pseudomonas stutzeri; Pseudomonas maltophilia; and Pseudomonas putrefaciens.

10. The microbe-based biofilm forming consortia as claimed in claim 1, wherein an antimicrobial secondary metabolite producing bacteria is Actinomycete sp. selected from Actinomyces viscosus; Actinomyces meyeri; Actinomyces naeslundii; Actinomyces odontolyticus; Actinomyces gerencseriae; Actinomyces neuii; Actinomyces turicensis; Actinomyces radingae; Actinomyces israelii; Actinomyces gerencseriae; Strptomyces hygroscopicus.

11. The formulation of microbe-based biofilm forming consortia as claimed in claim 7, wherein the Granular (GR) consortia comprises:
i. Microbe (A) Bacilli subtilis present in an amount of 1.0% to 30.0% w/w;
ii. Microbe (B) Pseudomonas aeruginosa present in an amount of 1.0% to 20.0% w/w;
iii. Microbe (C) Actinomycete present in an amount of 5.0% to 30.0% w/w;
iv. Carbon source in an amount of 10.0% to 60.0% w/w;
v. Wetting cum Emulsifying agent in an amount of 1.0% to 3.0 % w/w;
vi. Dispersing agent in an amount of 2.0% to 5.0% w/w;
vii. Solvent in an amount of 1.0% to 11.0% w/w.

12. The formulation of microbe-based biofilm forming consortia as claimed in claim 7, wherein the Controlled Release granule (CR-GR) consortia comprises:
i. Microbe (A) Bacillus subtilis present in an amount of 2.0% to 15.0% w/w;
ii. Microbe (B) Pseudomonas aeruginosa present in an amount of 5.0% to 20.0% w/w;
iii. Microbe (C) Actinomycete present in an amount of 5.0% to 30.0% w/w;
iv. Carbon source in an amount of 10.0% to 50.0% w/w;
v. Wetting cum Emulsifying agent in an amount of 1.0% to 3.0 % w/w;
vi. Dispersing agent in an amount of 2.0% to 5.0% w/w;
vii. Solvent in an amount of 1.0% to 11.0% w/w;
viii. Controlled release agent in an amount 5.0% to 25.0% w/w.

13. The microbe-based biofilm forming consortia as claimed in claims 5, 10 and 11, wherein the wetting cum emulsifying agent is selected from Mono C2-6 alkyl ether of a poly C2-4alkylene oxide block copolymer, condensation product of castor oil and polyC2-4alkylene oxide, alkoxylated castor oil is available under the trade name Agnique CSO-36, a mono- or di-ester of a C12-24 fatty acid and polyC2-4alkylene oxide, carboxylates, sulphates, sulphonates, alcohol ethoxylates, alkyl phenol ethoxylates, fatty acid ethoxylates, sorbitan esters, ethoxylated fats or oils, amine ethoxylates, phosphate esters, ethylene oxide - propylene oxide copolymers, fluorocarbons,alkyd-polyethylene glycol resin, polyalkylene glycol ether, apolyalkoxylated nonyl phenyl, alkoxylated primary alcohol, ethoxylated distyrylphenol, ethoxylated distyrylphenol sulphate, ethoxylated tristyrylphenol phosphate, tristyrylphenol phosphate ester, hydroxylated stearic acid polyalkylene glycol polymer, and their corresponding salts, alkyd-polyethylene glycol resin, polyalkylene glycol ether, ethoxylated distyrylphenol, ethoxylated distyrylphenol sulphate, ethoxylated tristyrylphenol phosphate, Alkyl phenol ethoxylate and ethoxylated tristrylphenol phosphate blend, tristyrylphenol phosphate ester, tristyrylphenol phosphate potassium salt, dodecysulfate sodium salt or a combination thereof.

14. The microbe-based biofilm forming consortia as claimed in claims 5, 10 and 11, wherein the dispersing agent is selected from Copolymer of propylene oxide (PO) and ethylene oxide (EO) and/or an ethoxylated tristyrene phenol, copolymer of PO and EO is alpha-butyl-omega-hydroxypoly (oxypropylene) block polymer with poly(oxyethylene), ethoxylated tristyrene phenol is alpha-[2,4,6-tris[1-(phenyl)ethyl] phenyl]-omega-hydroxy poly (oxyethylene, poly(oxy-1,2-ethanediyl)-alpha-C10-15alkyl-omega-hydroxy phosphate or sulphate and/or a C10-13 alkylbenzenesulfonic acid, tristyrylphenols, nonylphenols, dinonylphenol and octylphenols, styrylphenol polyethoxyester phosphate, alkoxylated C14-20 fatty amines or a combination thereof.

15. The microbe-based biofilm forming consortia as claimed in claims 5, 10 and 11, wherein a solvent is selected from Water, n-Octanol, n-butanol, Propylene glycol, polypropylene glycol, Fatty acid methyl ester, cyclohexane, xylene, mineral oil or kerosene, mixtures or substituted naphthalenes, mixtures of mono- and polyalkylated aromatics, dibutyl phthalate or dioctyl phthalate, ethylene glycol monomethyl or monoethyl ether, butyrolactone, octanol, castor oil, soybean oil, cottonseed oil, epoxidised coconut oil or soybean oil, aromatic hydrocarbons, dipropyleneglycol monomethylether, polypropylene glycol, polyoxyethylene polyoxypropylene glycols, polyoxypropylene polyoxyethylene glycols, diethyleneglycol, polyethylene glycol, methoxy polyethylene glycols; glycerol, methyl oleate, n-octanol, alkyl phosphates such as tri-n-butyl phosphate, propylene carbonate and isoparaffinic, tetrahydrofurfuryl alcohol, gamma-butyrolactone, N-methyl-2-pyrrolidone, tetramethylurea, dimethylsulfoxide, N,N-dimethylacetamide , Diacetone alcohol, Polybutene, Propylene carbonate, Dipropylene glycol isomer or a combination thereof.

16. The microbe-based biofilm forming consortia as claimed in claim 5, wherein a preservative agent is selected from 1,2 benzisothiazolin-3-one, 2-Methyl-2H-isothiazol-3-one, 5-chloro-2-methyl-4isothiazolin-3-one or sodium benzoate or benzoic acid or a combination thereof.

17. The microbe-based biofilm forming consortia as claimed in claim 5, wherein the stabilizer is selected from carboxylic acids (citric acid, acetic acid), orthophosphoric acid dodecyl benzene sulfonic acid and salt thereof.

18. The microbe-based biofilm forming consortia as claimed in claim 5, wherein the carrier is selected from Diatomaceous earth, attapulgite or zeolites, dolomite, limestone, silica, fly ash, hydrated lime, wheat flour, wood flour, ground wheat straw, cellulose and soy flour, bentonite, kaolin, attapulgite, diatomaceous earth, calcium carbonate, talc, muscovite mica, fused sodium potassium, aluminum silicate, perlite, talc and muscovite mica, urea, sulfur-coated urea, isobutylidene diurea, ammonium nitrate, ammonium sulfate, ammonium phosphate, triple super phosphate, phosphoric acid, potassium sulfate, potassium nitrate, potassium metaphosphate, potassium chloride, dipotassium carbonate, potassium oxide and a combination of these. Calcium, magnesium, sulfur, iron, manganese, copper, zinc; oxides, humic acid, Wood floor, Calcium silicate, Cellulose granules, Magnesium stearate or a combination thereof.

19. The microbe-based biofilm forming consortia as claimed in claim 5, wherein a coloring agent is selected from Crystal violet, Thalocyano dye chlorinated, Aerosol green FFB dye, Rodamine, Azo compound.

20. The microbe-based biofilm forming consortia as claimed in claims 5 and 11, wherein a controlled release agent is selected from Xanthan gum, PVK, carboxy methyl celluloses, polyvinyl alcohols, gelatin, sodium carboxy methyl cellulose, hydroxyl ethyl cellulose, Sodium Polyacrylate, modified starch, Paraffin wax, Polyvinyl acetate, Montan wax and vinyl acetate, Polyethylene Glycol, Cationic hydro soluble polymer, C4 alkylated Polyvinyl pyrrolidone or a combination thereof.

Dated this 10th Day of March, 2025